Introduction

In recent years, nanotechnology has been emerging as a rapidly growing field with numerous applications in science and technology for the purpose of manufacturing new materials (Savithramma et al. 2011). This technology is defined as the design, characterization, and application of structures, devices, and systems by controlling shape and size at nanometer scale level (1–100 nm). Nanoparticles are being viewed as fundamental building blocks of nanotechnology and defined as particles having one or more dimensions in the order of 100 nm or less. The most important and distinct property of nanoparticles is that they exhibit larger surface area-to-volume ratio. Among the nanoparticles studied so far, extensive research has been done on silver nanoparticles (Ag NPs) keeping in view of their potential bio-medical applications. Historically, silver metal has been used widely across the civilizations for different purposes. Many societies use silver as jewelry, ornamentation, and fine cutlery. Silver jewelry, wares, and cutlery were considered to impart health benefits to the users. There is an increasing use of silver as an efficacious antimicrobial agent in wound care products and medical devices (Kim et al. 2007; Shahverdi et al. 2007). Silver compounds are utilized as therapeutic modalities for wound management due to their biocidal activities toward a wide spectrum of microorganisms and their anti inflammatory effects (Tian et al. 2001; Liu et al. 2010) by contrast, the silver ion compounds may have cytotoxic effects and impair the wound healing process (Vanderplas et al. 2008). Production of nanoparticles can be achieved through different chemical methods like thermal decomposition of silver compounds (Plante et al. 2010), radiation assisted (Cheng et al. 2011) electro chemical (Hirsch et al. 2005) and recently green chemistry method (Ankamwar et al. 2005). Biological method, there is no need to use high pressure, energy, temperature, and toxic chemical that may have adverse effect in the medical applications (Nagajyothi et al. 2012). A number of living organisms are already well known to elaborate nanostructured composites such as cyanobacteria, bacteria, fungi, antinomycetes, biomolecules, and various plant materials such as Svensonia hyderobadensis (Lingarao and Savithramma 2012), Shorea tumbuggaia (Venkateswarlu et al. 2010) and Thespesia populnea (Bhumi et al. 2013), Ocimum tenuiflorum, Solanum tricobatum, Syzygium cumini, Centella asiatica, and Citrus sinensis (Logeswari et al. 2012). The potential of the plants as biological materials for the synthesis of nanoparticles is currently under exploitation. Hence the present study aimed to green synthesis of silver nanoparticles from stem bark extract of Cochlospermum religiosum (L) Alston and evaluation of their antimicrobial activity.

Cochlospermum religiosum (L) Alston is a sparsely branched small tree, belonging to the family Cochlospermaceae. It is commonly called as Yellow Silk Cotton, Buttercup Tree, and Torchwood Tree because of flowers are large, bright golden yellow, and seeds covered with silky hairs. Cochlospermum religiosum stem bark and root powder is traditionally used for fertility and ash of fruit mixed with coconut is used for the treatment of scabies (Goud et al. 2005). The gum of C. religiosum is also found to be an ingredient of unani medicine Qurs-e-Sartaan Kafoori which is used for Styptic, Antipyretic, Phthisis, Tuberculosis, Hectic fever, and Qurs-e-Suzak Cicatrizant, Diuretic, Gonorrhea. These formulations were found to possess good antibacterial and antifungal activity (Cecilie et al. 2005). Sasikala and Savithramma (2012a, b, c) studied the antimicrobial activity of biological synthesis of silver nanoparticles from leaves of C. religiosum and also studied the quantitative and quantification of phytochemicals (Sasikala and Savithramma 2012a, b, c; Sasikala et al. 2013a, b), in vitro propagation (Sasikala and Savithramma 2012a, b, c) and Histochemical (Sasikala et al. 2013a, b).

Materials and methods

All the chemicals and reagents used in the present study were of analytical grade. Silver nitrate was purchased from Sigma-Aldrich chemicals. The glassware were washed in dilute nitric acid and thoroughly washed with double distilled water and dried in hot air oven.

Preparation of plant extract

Cochlospermum religiosum stem bark was collected from Tirumala hills of Andhra Pradesh, India. The barks were washed thoroughly thrice with distilled water and were shade dried for 10 days. The fine powder was obtained from dried leaves using kitchen blender. The bark powder was sterilized at 121 °C for 5 min. 5 g of powder were taken into 250 ml conical flask and added 100 ml of sterile distilled water and boiled for 15 min at 100 °C. Then the bark extract was collected in separate conical flask by standard filtration method.

Synthesis of silver nanoparticles

1 mM AgNO3 solution was prepared and stored in amber color bottle. The bark extract was added to 1 mM AgNO3 solution. The color change of the solution from yellow to brown indicated the silver nanoparticles were synthesized from the bark for the characterization and antimicrobial activity.

UV–Vis spectra analysis

The reduction of pure silver ions was monitored by measuring the UV–Vis spectrum of the reaction medium at 5 h. After diluting, a small aliquot analysis was done using UV–Vis spectrophotometer UV-2450 (Shimadzu).

X-ray diffraction (XRD) analysis

The practice size and nature of the silver nanoparticle were determined using XRD. This was carried out using Shimadzu XRD-6000/6100 model with 30 kV, 30 mA with Cuk ∝ radians at 2θ angle. X-ray powder diffraction is a rapid analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell dimensions. The analyzed material is finely ground—and average bulk composition is determined. The particle or grain size of the particles on the silver nanoparticles was determined using Debye Sherrer’s equation,

$$ {\text{D }} = {\text{ k}}\lambda /\beta ({\text{Cos}}\theta ). $$

Fourier transform-infrared spectrophotometic (FT-IR) analysis

The functional group of silver nanoparticles was identified by FT-IR using (Thermo Nicolet-nexus 670 spectrometer of resolution 4 cm−) one drop of sample was placed between the plates of sodium chloride. The drop forms a thin film between the plate sodium chloride is transparent to infrared light.

SEM analysis of silver nanoparticles

Scanning electron microscope (SEM) analysis was carried out using Hitachi S-4500 SEM Machine. Thin films of the sample were prepared on a carbon-coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid was allowed to dry.

Energy dispersion spectrophotometric (EDS) measurements

In order to carryout EDAX analysis, the drop of bark extract with reduced silver nanoparticles was dried on coated with carbon film and performed on Hitachi S-3400N SEM instrument equipped with thermo EDAX attachments.

Atomic force microscopic (AFM) measurements

The silver nanoparticles extracted through above protocol were visualized with an atomic force microscope. A thin film of the sample was prepared on a glass on the slide was allowed to dry for 5 min, the slides were then scanned with the AFM (Nano Surf® AG, Switzerland, and Product: BTO 2089, BRO).

Evaluation of the antimicrobial activity of silver nanoparticles synthesized using stem bark extract of Cochlospermum religiosum

Microorganisms

Pure cultures of E. coli, Pseudomonas aeruginosa, Bacillus subtilis, Proteus vulgaris, and Staphylococcus species of bacteria and Fusarium oxysporum, Curvularia lunata, Rhizopus arrhizus, Aspergillus niger, and Aspergillus flavus species of fungi were procured from the Department of Microbiology of Sri Venkateswara Institute of Medical Science (SVIMS). The experiments of antimicrobial activity were carried out in the Department of Microbiology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.

Antibacterial activity

The antibacterial activity of SNPs was carried out by disk diffusion method (Bauer et al. 1966). Nutrient agar medium plates were prepared, sterilized, and solidified. After solidification bacterial cultures were swabbed on these plates. The sterile disks were dipped in silver nanoparticles solution (10 μg/ml) and placed in the nutrient agar plate and kept for incubation at 37 °C for 24 h. Zones of inhibition for control, SNPs and silver nitrate were measured. The experiments were repeated thrice and mean values of zone diameter were measured with the help of MIC Scale, and the results were tabulated.

Antifungal activity

Potato dextrose agar plates were prepared, sterilized, and solidified, after solidification fungal cultures were swabbed on these plates. The sterile disks were dipped in silver nanoparticles solution (10 μg/ml) and placed in the agar plate and kept for incubation for 7 days. After 7 days, zone of inhibition was measured with the help of MIC Scale, and the results were tabulated.

Results and discussion

The time of addition of plant extract into the silver nitrate solution was considered as the start of the reaction. It is well known that the silver nanoparticles exhibit yellowish brown color in aqueous solution due to excitation of Surface Plasmon Resonance. Cochlospermum religiosum bark extract was mixed in the aqueous solution of silver nitrate, initially the color changed from light yellowish to dark brown color (Fig. 1) due to the reduction of silver ion, similar results were observed in various plants studied by Lingarao et al. (2013); Ankanna et al. (2010). Formation and stability of silver nanoparticles in aqueous colloidal solution are confirmed using UV–Vis spectral analysis. The UV–Vis spectrum showed maximum absorbance at 445 nm (Fig. 2), which increased with time of incubation of silver nitrate with the plants extract. The broadening of peak indicated that the particles are poly dispersed. It has been reported earlier that silver has absorbance at round 430 nm (Nestor et al. 2008). The presence of the secondary metabolites in plant extract may be responsible for the reduction of silver and synthesis of nanoparticles. The biogenic route is the energy (or) electron released during Glycolysis (photosynthesis) for conversion of NAD to NADH led to transformation of Ag(NO3)2 to form nanoparticles and another mechanism is releasing of an electron when formation of ascorbate radicals from ascorbate reduces the silver ions,

$$ {\text{AgNO}}_{ 3} \to {\text{Ag }} + {\text{ NO}}_{ 3} $$
$$ {\text{NAD}}^{ + } + {\text{ e}} \to {\text{NAD}} $$
$$ {\text{NAD }} + {\text{ H}}^{ + } \to {\text{NADH }} + {\text{ e}}^{ - } $$
$$ {\text{e}}^{ - } + {\text{ Ag}}^{ + } \to {\text{Ag}}^{0} . $$
Fig. 1
figure 1

a Plant extract, b plant extract with silver nitrate (SNPs) the color change of stembark extract of C. religiosum

Full size image
Fig. 2
figure 2

UV–Visible spectroscopic micrograph showing the characteristic absorbance of C. religiosum stem bark-synthesized silver nanoparticles at 445 nm

Full size image

The XRD pattern showed number of Bragg reflections that may be indexed on the basis of face-centered cubic structure of silver. The XRD analysis is confirmed that the silver particles formed in the experiments were in the form of nanocrystals, as evidenced by the peaks at 2θ values of 38.28° and 48.04° corresponding to (111) and (200) Bragg reflections, respectively, in Fig. 3 are through to be related to crystalline and amorphous organic phases. It was found that the average size form XRD data and using the Debye–Scherrer equation was approximately 28.64 nm.

Fig. 3
figure 3

XRD micrograph showing the Bragg’s reflections corresponds to the face-centered cubic (FCC) structure of SNPs synthesized using the stembark of C. religiosum

Full size image

The SEM analysis was used to determine the structure of the reaction products that were formed. SEM image has showed individual silver particles as well as number of aggregates, SEM images of SNPs derived from the aqueous bark extracts of C. religiosum showed the particles in spherical shape and size ranged from 20 to 35 nm (Fig. 4). The morphology of the SNPs was predominantly spherical and they appear to be monodispersed. Further, analysis of the silver particles by energy dispersive spectroscopy confirmed the presence of the signal characteristic of silver (Fig. 5). All the peaks of Ag are observed and are assigned. Peaks of Ag are from the grid used and the peaks of S, P, and N correspond to the protein capping over the AgNPs. EDAX information has given the various elements along with SNPs of bark extracts of C. religiosum was identified elements like C, O, Mg, Si, Ag, and Al with different percentages.

Fig. 4
figure 4

SEM micrograph revealing the presence of the silver nanoparticles (agglomerated) of synthesized using C. religiosum Stembark

Full size image
Fig. 5
figure 5

EDS spectrum evidencing the elemental presence of silver along with calcium, magnesium silicon, oxygen, and carbon

Full size image

AFM analysis the SNPs were clearly distinguishable owing to their size difference. An AFM image has given average sizes of SNPs of C. religiosum is 45 nm with three dimensional structures. SNPs attached with one another and look like a cluster in an area of 15 μm with rod shape in 3D view (Fig. 6). The physicochemical properties of nanoparticles differ dramatically from fine particles of the same composition (Geraci and Castranova 2010).

Fig. 6
figure 6

AFM image detailing the topographical information of SNPS synthesized using the stem bark extract of Cochlospermum religiosum

Full size image

The FTIR spectra Fig. 7 indicate different functional groups at various positions. The vibrational bands correspond to the bonds such as aminuteso (N–H), –C=C (alkene), and C–Cl (Halogens) which were in the region range of 1,194–3,315 cm−1. The most wide spectrum absorption was observed at 3,315  cm−1, it can be attributed to the stretching vibrations of aminuteso (N–H) (Maliszewska and Sadowski 2009) and absorption peaks observed at 1,636 cm−1 can be attributed to the stretching vibration of –C=C (alkene) (Rajasekharreddy et al. 2010). This FT-IR spectroscopic study confirmed that the carbonyl group of amino acid residues has a strong binding ability with silver, suggesting the formation of a layer covering silver nanoparticles and acting as a capping agent to prevent agglomeration and provide stability to the medium. These results confirm the presence of possible proteins as reducing stabilizing agents.

Fig. 7
figure 7

FT-IR spectra representing the functional groups associated with the reduction and stabilization of Cochlospermum religiosum stembark-mediated silver nanoparticles

Full size image

The antimicrobial activity of silver nanoparticles was carried out against various pathogenic microbes such as gram negative and gram positive bacteria of Staphylococcus, E. coli, Proteus vulgaris, Pseudomonas aeruginosa, and Bacillus subtilis, fungal species of Aspergillus niger, Aspergillus flavus, Fusarium, Curvularia, and Rhizopus using disk diffusion method. The extraction without silver nanoparticles served as control. Bark aqueous extract of C. religiosum showed broad spectrum of antimicrobial activity. The diameter of inhibition zone around each disk with SNPs is measured and each disk contains of 20 μl of SNPs solution (Table 1). The SNPs of bark extract of C. religiosum showed highest antibacterial activity against Staphylococcus, followed by Pseudomonas, E. coli, Bacillus, and lowest activity toward Proteus and maximum inhibition zone was observed against fungal species of Aspergillus flavus followed by Rhizopus, Fusarium Curvularia, and minimum inhibition zone was observed against Aspergillus niger (Figs. 8, 9).

Table 1 Antimicrobial activity of stem bark aqueous extract and silver nanoparticles of C. religiosum
Full size table
Fig. 8
figure 8

Antimicrobial activity of biologically synthesized SNPs of stembark against a Bacillus, b E. coli, c Proteus, d Pseudomonas, e Staphylococcus, f Aspergillus flavus, g Fusarium, h Curvularia lunata, i Rhizopus, j Aspergillus niger, 1 leaf extract, 2 leaf SNPs, 3 Silver nitrate, 4 Gentamycin/Nystatin

Full size image
Fig. 9
figure 9

Antimicrobial activity of stem bark aqueous extract and silver nanoparticles synthesized using C. religiosum

Full size image

It is interesting to note that the SNPs synthesized via green route are highly toxic toward bacterial strains when compared to fungal strains. Silver ions have been demonstrated to interact with the protein and possibly phospholipids associated with the proton pump of bacterial membranes (Savithramma et al. 2012). These results in a collapse of membrane proton gradient causing a disruption of many of the mechanisms of cellular metabolism and hence cell death (Dibrov et al. 2002). Silver ions interact with a wide range of molecules processes within a micro organism resulting in a range of effects from inhibition of growth and loss of ineffectiveness. The mechanism depends on both the concentration of silver ions present and the sensitivity of the microbial species to silver. The spectrum of activity is very wide and the development of resistance relatively law (Cooper 2004). The use of plant extracts is effective against various microorganisms including plant pathogens (Mishra et al. 2007).

Conclusion

Stem bark extract of Cochlospermum religiosum was proved to be one of the potential sources to produce stable and well-defined nanoscale silver particles. Using medicinal plants and plant parts for the synthesis of metallic nanoparticles, silver in particular, has logical background for their use in therapeutic applications. The conjunctive effect of both silver nanoparticles and the medicinal biomolecules present on the surface of the silver nanoparticles was significant against an array of microbes as the bacterial and fungal strains are getting resistance to traditional and standardized drugs.